The blood-brain barrier (BBB) represents a significant bottleneck for the delivery of therapeutics to the central nervous system. In recent years, the promise of coopting BBB receptor-mediated transport systems for brain drug delivery has increased in large part due to the discovery and engineering of BBB-targeting antibodies. Here we describe an innovative screening platform for identification of new BBB targeting molecules from a class of lamprey antigen recognition proteins known as variable lymphocyte receptors (VLRs). Lamprey were immunized with murine brain microvessel plasma membranes, and the resultant repertoire cloned into the yeast surface display system. The library was screened via a unique workflow that identified 16 VLR clones that target extracellular epitopes of in vivo-relevant BBB membrane proteins. Of these, three lead VLR candidates, VLR-Fc-11, VLR-Fc-30, and VLR-Fc-46 selectively target the brain vasculature and traffic within brain microvascular endothelial cells after intravenous administration in mice, with VLR-Fc-30 being confirmed as trafficking into the brain parenchyma. Epitope characterization indicates that the VLRs, in part, recognize sialylated glycostructures. These promising new targeting molecules have the potential for brain targeting and drug delivery with improved brain vascular specificity.
© 2022. The Author(s).

Drug delivery across the blood-brain barrier (BBB) remains a significant obstacle for the development of neurological disease therapies. The low penetration of blood-borne therapeutics into the brain can oftentimes be attributed to the restrictive nature of the brain microvascular endothelial cells (BMECs) that comprise the BBB. One strategy beginning to be successfully leveraged is the use of endogenous receptor-mediated transcytosis (RMT) systems as a means to shuttle a targeted therapeutic into the brain. Limitations of known RMT targets and their cognate targeting reagents include brain specificity, brain uptake levels, and off-target effects, driving the search for new and potentially improved brain targeting reagent-RMT pairs. To this end, we deployed human-induced pluripotent stem cell (iPSC)-derived BMEC-like cells as a model BBB substrate on which to mine for new RMT-targeting antibody pairs. A nonimmune, human single-chain variable fragment (scFv) phage display library was screened for binding, internalization, and transcytosis across iPSC-derived BMECs. Lead candidates exhibited binding and internalization into BMECs as well as binding to both human and mouse BBB in brain tissue sections. Antibodies targeted the murine BBB after intravenous administration with one particular clone, 46.1-scFv, exhibiting a 26-fold increase in brain accumulation (8.1 nM). Moreover, clone 46.1-scFv was found to associate with postvascular, parenchymal cells, indicating its successful receptor-mediated transport across the BBB. Such a new BBB targeting ligand could enhance the transport of therapeutic molecules into the brain.
© 2020 Federation of American Societies for Experimental Biology.

Neuritic plaques, a pathological hallmark in Alzheimer's disease (AD) brains, comprise extracellular aggregates of amyloid-beta (Aβ) peptide and degenerating neurites that accumulate autolysosomes. We found that, in the brains of patients with AD and in AD mouse models, Aβ plaque-associated Olig2- and NG2-expressing oligodendrocyte progenitor cells (OPCs), but not astrocytes, microglia, or oligodendrocytes, exhibit a senescence-like phenotype characterized by the upregulation of p21/CDKN1A, p16/INK4/CDKN2A proteins, and senescence-associated β-galactosidase activity. Molecular interrogation of the Aβ plaque environment revealed elevated levels of transcripts encoding proteins involved in OPC function, replicative senescence, and inflammation. Direct exposure of cultured OPCs to aggregating Aβ triggered cell senescence. Senolytic treatment of AD mice selectively removed senescent cells from the plaque environment, reduced neuroinflammation, lessened Aβ load, and ameliorated cognitive deficits. Our findings suggest a role for Aβ-induced OPC cell senescence in neuroinflammation and cognitive deficits in AD, and a potential therapeutic benefit of senolytic treatments.

Globoid cell leukodystrophy (GLD), or Krabbe disease, is an inherited, neurologic disorder that results from deficiency of a lysosomal enzyme, galactosylceramidase. Most commonly, deficits of galactosylceramidase result in widespread central and peripheral nervous system demyelination and death in affected infants typically by 2 years of age. Hematopoietic stem-cell transplantation is the current standard of care in children diagnosed prior to symptom onset. However, disease correction is incomplete. Herein, the first adeno-associated virus (AAV) gene therapy experiments are presented in a naturally occurring canine model of GLD that closely recapitulates the clinical disease progression, neuropathological alterations, and biochemical abnormalities observed in human patients. Adapted from studies in twitcher mice, GLD dogs were treated by combination intravenous and intracerebroventricular injections of AAVrh10 to target both the peripheral and central nervous systems. Combination of intravenous and intracerebroventricular AAV gene therapy had a clear dose response and resulted in delayed onset of clinical signs, extended life-span, correction of biochemical defects, and attenuation of neuropathology. For the first time, therapeutic effect has been established in the canine model of GLD by targeting both peripheral and central nervous system impairments with potential clinical implications for GLD patients.

Adult neurogenesis persists in the rodent dentate gyrus and is stimulated by chronic treatment with conventional antidepressants through BDNF/TrkB signaling. Ketamine in low doses produces both rapid and sustained antidepressant effects in patients. Previous studies have shed light on post-transcriptional synaptic NMDAR mediated mechanisms underlying the acute effect, but how ketamine acts at the cellular level to sustain this anti-depressive function for prolonged periods remains unclear. Here we report that ketamine accelerates differentiation of doublecortin-positive adult hippocampal neural progenitors into functionally mature neurons. This process requires TrkB-dependent ERK pathway activation. Genetic ablation of TrkB in neural stem/progenitor cells, or pharmacologic disruption of ERK signaling, or inhibition of adult neurogenesis, each blocks the ketamine-induced behavioral responses. Conversely, enhanced ERK activity via Nf1 gene deletion extends the response and rescues both neurogenic and behavioral deficits in mice lacking TrkB. Thus, TrkB-dependent neuronal differentiation is involved in the sustained antidepressant effects of ketamine.